Disorders of the central nervous system (CNS) include a number and variety of conditions, such as neurodegenerative diseases (e.g. Alzheimer's and Parkinson's), acute brain injury (e.g. stroke, head trauma, cerebral palsy) and neurological dysfunction (e.g. depression, epilepsy, schizophrenia). As the elderly population grows, neurodegenerative disease becomes an increasingly important concern, as the risk for many of these disorders increases with age. These neurodegenerative diseases, which include Alzheimer's disease (AD), multiple sclerosis (MS), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD), have been linked to the degeneration of neural cells in identified locations of the CNS, resulting in an inability of these cells or the relevant brain region to carry out their intended function.
Treatment for CNS disorders via the administration of pharmaceutical compounds has drawbacks, including the limited range of drugs capable of crossing the blood-brain barrier and the drug-tolerance that develops in patients receiving long-term treatment. For example, Parkinson's patients treated with levodopa (L-dopa), a dopamine precursor that is able to cross the blood-brain barrier become tolerant to the effects of L-dopa, and steadily increasing dosages are needed to maintain its effects. In addition, there are a number of side effects associated with L-dopa, such as increased and uncontrollable movement.
Over 1.5 million people in the United States suffer from Parkinson's disease (PD). Once pharmacological treatment for PD is exhausted, patient can only turn to surgical interventions. Current interventions focus on containing PD symptoms, but it is imperative to attempt to reverse the damage of the disease. Such restoration may be possible through transplantation of neural progenitor cells.
Grafting of fetal neural tissue has been applied to the treatment of neurological diseases such as Parkinson's disease. Fetal neural grafts may avert the need for constant drug administration, and also for drug delivery systems designed to circumvent the blood-brain barrier. However, the cells used for transplantation can induce an immune reaction in the host recipient. In addition, the cells must be at a stage of development where they are able to form normal neural connections with neighboring cells.
Grafting also offers a therapeutic approach to demyelinating diseases, such as multiple sclerosis (MS). In both human demyelinating diseases and rodent models there is substantial evidence that demyelinated neurons are capable of remyelination in vivo. In MS, for example, it appears that there are often cycles of de- and remyelination. Exogenously applied cells have been shown to be capable of remyelinating demyelinated axons in a number of experimental conditions (See Freidman et al., Brain Research, 378:142-146, 1986; Raine, et al., Laboratory Investigation 59:467-476, 1988). Success has been shown using dissociated glial cell suspensions prepared from spinal cords Duncan et al., J. Neurocytology, 17:351-360 (1988); Schwann cell cultures prepared from sciatic nerve (Bunge et al., 1992, WO 92/03536; Blakemore and Crang, J. Neurol. Sci., 70:207-223, 1985); cultures from dissociated brain tissue (Blakemore and Crang, Dev. Neurosci. 10:1-11, 1988); oligodendrocyte precursor cells (Gumpel et al., Dev. Neurosci. 11:132-139, 1989); O-2A cells (Wolswijk et al., Development 109:691-608, 1990; Raff et al., Nature 3030:390-396, 1983; Hardy et al., Development 111:1061-1080, 1991); and immortalized O-2A cell lines (Almazan and McKay, Brain Res. 579:234-245, 1992).
O-2A cells are glial progenitor cells which give rise in vitro only to oligodendrocytes and type II astrocytes. Cells immunopositive in vivo for the O-2A phenotype have been shown to successfully remyelinate demyelinated neurons in vivo, (Godfraind et al., J. Cell Biol. 109:2405-2416, 1989). Injection of a large number of O-2A cells is required to adequately remyelinate all targeted neurons in vivo. Although O-2A progenitor cells can be grown in culture, they are capable of only a limited number of divisions (Raff Science 243:1450-1455, 1989). In addition, the isolation technique employs a low yield source (optic nerve) and requires a number of purification steps.
Various approaches to neurotransplantation have been developed to ameliorate neurological disease, including the grafting of neurons from the adult PNS to produce dopamine (Notter, et al., Cell Tissue Research 244:69-76, 1986), transplantation of monoamine-containing cells isolated from adult rat pineal gland and adrenal medulla into rat frontal cortex to alleviate learned helplessness, a form of depression (U.S. Pat. No. 4,980,174); grafting of chromaffin cells and adrenal medullary into the brain stem or spinal cord of rats to produce analgesia when the implanted tissue or cell was induced to release catecholamines (U.S. Pat. No. 4,753,635). Adrenal cells, however, do not obtain a normal neural phenotype upon grafting into the CNS, and are therefore of limited use for transplants where synaptic connections must be formed.
Another approach to neurotransplantation involves the use of genetically modified cells. Using this method, a foreign gene or transgene is introduced into a cell to allow the cell to express the gene. Cells modified to contain the transferred gene can be transplanted to the site of neurodegeneration, and provide products such as neurotransmitters and growth factors (Rosenberg, et al., Science 242:1575-1578, 1988) which may function to alleviate some of the symptoms of degeneration. Genetically modified cells have been used in neurological tissue grafting in order to replace lost cells. For example, fibroblasts have been genetically modified with a retroviral vector containing a cDNA for tyrosine hydroxylase, which allows them to produce dopamine, and implanted into animal models of Parkinson's Disease (U.S. Pat. No. 5,082,670). However, there remains a risk of inducing an immune reaction using currently available cell lines, and these cells may not achieve normal neuronal connections within the host tissue.
While attempts have been made to propagate neural progenitor cells for use in neurotransplantation and for drug screening, these efforts have met with limited success. Neurobasal medium has allowed for fast doubling times of cultured neural progenitor cells, but these doubling times are observed for about one month, after which the cells differentiate and lose their progenitor phenotype. Typically, with the most optimal culture conditions, neural progenitor cells will survive for only about 10 passages in culture. In addition, only about 1-2% of neural progenitor cells survive cryopreservation. Moreover, current efforts to maintain neural progenitor cells in vitro require the use of a feeder layer and/or introduce animal components. Even with use of a feeder layer, neural progenitor cells have been maintained for only about 6 months. For clinical applications, it is desirable to obtain and maintain human neural progenitor cells that are free of animal components and do not require the use of a feeder layer.
There remains a need for a large quantities of undifferentiated neural progenitor cells and pluripotent stem cells for transplantation and for drug screening, particularly for human progenitor and stem cells. A need also exists for neural progenitor cells that are capable of long-term proliferation in vitro and that are amenable to controlled differentiation and/or genetic modification. In particular, there is a need for methods of maintaining and propagating neural progenitor cells for extended periods of time, and for methods that optimize yield following cryopreservation.